Pure drug samples
Pure and active form of LAM (99.4%), EVZ (99.4%), and TDF (99.3%) were procured as gift samples from Aurobindo Pharma, Hyderabad.
Formulation
Film-coated tablet dosage forms (Symfi Lo is a fixed-dose combination product containing 400 mg of EVZ, 300 mg of LAM, and 300 mg of TDF) were purchased from local pharmacy in Hyderabad.
Chemicals and reagents
All HPLC grade and analytical grade solvents were purchased by local distributor of Merck India.
Instrument specifications
The method was done by using WATERS HPLC (2695model, PDA detector, and the Empower 2 solution software). In addition, an analytical balance (Denver instrument, TB-215D), a digital pH meter (MEZARIT), and water (Milli-Q) were used. Forty-five micrometers povidone filters were used to filter all the solutions and the solvents before introduced into the instrument.
Chromatographic method conditions
An efficient and appropriate separation of the three analytes was done with Zorbax eclipse XDB-Phenyl (250 × 4.6mm, 5μ) column, using a mobile phase consist of methanol: buffer (0.1% v/v formic in water) (73:27 v/v) at a flow rate of 1mL/min and a detection wavelength of 260.0 nm. Equal ratio of acetonitrile and water was used as diluents.
Preparation of standard solution
One hundred milligrams of LAM, 100.0mg of TDF, and 133.3mg of EVZ pure powders were accurately weighed and dissolved with diluent in a 100-mL volumetric flask. One milliliter of the above solution was diluted to 10mL. Three milliliters of the resultant solution was further diluted to 10mL to get a concentration of 30.0μg/mL, 40.0μg/mL, and 30.0μg/mL for LAM, EVZ, and TDF respectively.
Preparation of sample solution
The crushed tablet powder equivalent to 100mg of LAM, 100mg of TDF, and 133.3mg of EVZ pure powders was accurately weighed and dissolved with diluent in 100-mL volumetric flask. One milliliter of the above solution was diluted to 10mL. Three milliliters of the resultant solution was further diluted to 10mL to get a concentration of 30.0μg/mL, 40.0μg/mL, and 30.0μg/mL for LAM, EVZ, and TDF respectively.
Method validation
Validation is written evidence that provides assurance of the method or process with high degree level. To validate analytical method, Q2R1 guidelines of the ICH were taken into consideration.
System suitability test
The system suitability test of the present method was established by injecting standard solution in 6 consecutive injections, and the parameters such as percentage relative standard deviation (%RSD), tailing factor (T), resolution (R), and number of theoretical plates (N) were assessed for the chromatograms thus attained.
Linearity
The linearity of an analytical method signifies the direct proportional relationship between experimental results and the given concentrations. It was established for the solutions having concentrations ranging from 15.0 to 45.0 μg/mL of both LAM and TDF and 20.0 to 60.0μg/mL of EVZ. A calibration curve was plotted between concentration and peak area to determine regression coefficient (R2).
Precision
The precision of the method expresses the close agreement between the observed responses of homogenous samples on multiple sampling at identical conditions. In general, it can be performed on the same day and on different consecutive days represented as intra-day (repeatability) precision and inter-day precision (intermediate precision) respectively. Repeatability was assessed by injecting standard solution for five times in a single day, and intermediate precision was assessed by injecting same standard solution for two times per day for three successive continuous days. %RSD values were assessed for peak areas attained in chromatograms.
Accuracy
Substantially, the accuracy was validated by percentage recovery studies where specified amount of sample solution was spiked into standard solution. In general, spiking can be done at 50, 100, and 150% level concentrations of standard solution. Each level solution was injected for three times. The % mean recovery of sample at each spiked level was calculated.
Specificity
Specificity is the ability of the analytical method to assess the analyte of interest in the presence of other substances including degradation products and excipients without interferences. It was performed by introducing consecutive injections of placebo, blank, standard, and placebo mixed with standard. The obtained chromatograms were examined for interference from the peaks of other substances with peaks of analytes to be determined.
Sensitivity
The LOQ and LOD were reckoned by using the following formulae:
$$ \mathrm{LOD}=3\times \upsigma /S $$
$$ \mathrm{LOQ}=10\times \upsigma /S $$
where σ is the SD of intercept and S is the slope of the linear curve.
Robustness
Method’s robustness was assessed by changing the optimized conditions of the method to a little extent intentionally. In this case, optimized conditions like ratio of the mobile phase (± 1mL), flow rate of mobile phase (± 0.1 mL/min), and detection wavelength (± 2nm) were intentionally altered to a little extent.
Forced degradation studies
In forced degradation (FD) studies, chemical stability of the analyte can be assessed in the presence of more intensive conditions like acid and alkali hydrolysis, oxidative degradation, and thermal and photo stability as provided by ICH quality guidelines.
Acid and alkali hydrolysis
Ten milliliters of standard stock solution was mixed with 2mL of 0.1N HCl and 2mL of 0.1N NaOH separately, reflux the prepared solutions for 2 h at 70°C, and kept it aside at room temperature for 24 h. The resultant solutions were neutralized and further diluted in such a way to get a concentration of 30μg/mL, 40μg/mL, and 30μg/mL for LAM, EVZ, and TDF respectively.
Oxidative degradation
Ten milliliters of standard stock solution was mixed with 2mL of 3% hydrogen peroxide and reflux the prepared solutions for 2h at 70 °C, and kept it aside for 24 h and further diluted in such a way to get a concentration of 30μg/mL, 40μg/mL, and 30 μg/mL for LAM, EVZ, and TDF respectively.
Thermal degradation
The standard stock solution was placed in hot air oven at 80°C/75% relative humidity for 24 h. One milliliter of the above exposed solution was further diluted in such a way to get a concentration of 30μg/mL, 40μg/mL, and 30μg/mL for LAM, EVZ, and TDF respectively.
Photo degradation
The standard stock solution was in ultraviolet chamber at 254.0 nm for 24 h. One milliliter of above exposed solution was further diluted in such a way to get a concentration of 30μg/mL, 40μg/mL, and 30μg/mL for LAM, EVZ, and TDF respectively.
The above mentioned FD solutions were injected and computed for the percentage degradation of LAM, EVZ, and TDF. As per most of the researcher’s suggestions, the considerable degradation of analyte is around 20% for the validation of stability-indicating HPLC method.
Assay
The assays of the LAM, EVZ, and TDF in commercial tablets were determined by injecting sample solution.